The de novo synthesis of fatty acids occurs in two distinct cellular compartments. Palmitate (C16:0) is synthesized in the cytoplasm by acetyI-CoA carboxylasel and fatty acid synthase. The vast majority of palmitate synthesized is then elongated to longer chain fatty acids in the endoplasmic reticulum via four sequential reactions: condensation, reduction, dehydration, and a final reduction to form the elongated fatty acyI-CoA. The objectives of this application are to identify and characterize the function of three enzymes involved in microsomal fatty acid elongation. First, we will investigate the in vivo function of the long chain fatty acyl elongase (LCE), the initial condensing enzyme required for the elongation of long chain fatty acids in vitro. We will use LCE knockout mice to determine whether LCE is required for the elongation of palmitate in vivo and whether its activity is required for normal lipid homeostasis in liver. Additional studies will determine whether the lack of LCE activity attenuates or prevents the development of obesity and diabetes in mice. Second, we have identified a novel condensing enzyme, designated ELOVL7. Unlike other condensing enzymes, ELOVL7 is not expressed in liver, but is highly expressed in intestine. We will determine the fatty acid substrate specificity of ELOVL7, and characterize the biologic function of ELOVL7 in intestine using knockout mice. Preliminary data suggests that ELOVL7 elongates long chain saturated and monounsaturated fatty acids. Therefore, we will determine whether ELOVL7 is required for the normal synthesis of lipids that have long chain saturated fatty acid moieties, such as ceramide and sphingolipids. Third, we will identify and characterize the microsomal 3-hydroxystearoyI-CoA dehydratase. This enzyme catalyzes the third reaction in microsomal fatty acid elongation, and is the only unidentified enzyme in this pathway. Studies, both in vitro and in vivo, will focus on characterizing the activity and regulation of this enzyme. The studies proposed in this application will provide a comprehensive characterization of three mammalian microsomal fatty acid elongation enzymes and will determine whether enzymes involved in microsomal fatty acid elongation are potential targets for treating common diseases such as hypertriglyceridemia, obesity, and insulin-resistant diabetes.